This proposal is concerned with the cytoplasmic determinants in the Ascidian egg that appear to regulate the development of three histospecific enzymes in the embryo: muscle acetylcholinesterase, endodermal alkaline phosphatase, and brain melanocyte tyrosinase. The studies are directed at determining the nature of the agents, their mechanism of action, and how their localization and segregation are controlled. Determinant movements will be studied in relation to changes in the actin and tubulin cytoskeleton of early embryos and in relation to changes in lectin-binding sites on the egg plasma membrane. Combined blastomere isolation and culturing techniques and two-dimensional gel electrophoresis will be used to find other protein markers of lineage-specific cell differentiation. The nature of the biological "clock" mechanism which times differentiation in the embryos will be examined, especialy to determine whether it is based on cytoplasmic or nuclear cycles. A specific antibody to ascidian larval acetylcholinesterase will be used to verify time of enzyme synthesis and to show by in vitro translation when enzyme messenger RNA first occurs in development. Similar techniques will be employed to show that alkaline phosphatase egg cytoplasmic determinant is a preformed maternal mRNA for the enzyme. A biological assay system based on microinjection of materials into non-lineage blastomeres of the early embryo will be divised to use in isolating and characterizing the acetylcholinesterase determinant and other determinants. Certain of the ascidian morphogenetic determinants are presumed to represent a class or group of substances that regulate gene control of cell differentiation in all embryos. What emerges from the studies in this proposal should have wide relevance to any consideration of gene regulatory mechanisms in normal and abnormal development.